We isolated total RNA of 461 subjects from serum samples using Qiagen miRNeasy Serum/Plasma extraction kit and QIAcube automation. All samples were quantified using the Nanodrop spectrophotometer prior to plating with the RNA concentration 30.97 ± 20.97 ng/μl (see Additional file 1 : Fig. S1). We built the small RNA-Seq libraries by Norgen Biotek Small RNA Library Prep Kit and sequenced on the Illumina NextSeq 500 platform at 51 bp single end reads. The sequencing data was deposited in the Gene Expression Omnibus with the accession number GSE134897 [13 (link)]. COMPSRA was employed to evaluate the read quality and trim adapters [22 (link)]. Sequencing reads with quality score lower than 20 were removed. The qualified reads were aligned to human genome hg38 by STAR (v2.7.10b) [23 (link)] and miRNAs were annotated by COMPSRA on the basis of miRbase [24 (link)].
Small rna library prep kit
Manufactured by Illumina
Sourced in Canada
The Small RNA Library Prep Kit is a laboratory instrument designed for the preparation of small RNA samples for sequencing. The kit provides a standardized workflow for the isolation, purification, and library construction of small RNA molecules, such as microRNAs, from various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 platform, by Illumina (5 mentions)
Hiseq 2000, by Illumina (4 mentions)
Mirneasy serum plasma extraction kit, by Qiagen (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Single cell rna purification kit, by Norgen Biotek (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Novaseq, by Illumina (1 mentions)
Mirnaeasy serum plasma extraction kit, by Qiagen (1 mentions)
13 protocols using small rna library prep kit
1
Serum Small RNA Sequencing Protocol
2
MicroRNA Sequencing and Analysis Protocol
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MicroRNAs were sequenced using the NEBNext Small RNA library prep kit on an Illumina HiSeq 2000. The short reads were trimmed of adapters with Cutadapt [14 ]. Trimmed microRNA sequences greater than 17 nucleotides in length were then aligned to the reference genome and miRBase reference sequences using Bowtie [15 (link)]. Known microRNA expression and novel microRNA prediction and quantification were performed with miRDeep2 [16 (link)], using the CAP-miRSeq analysis pipeline [17 (link)]. Unsupervised hierarchical clustering was performed using the Pearson correlation method. ComiR, a computational tool for combinatorial microRNA target prediction was used to identify molecular pathways regulated by microRNAs that were differentially expressed between diseased and non-diseased palmar fascia [18 (link), 19 (link)]. The Database for Annotation and Visualization and Integrated Discovery v6.7 (DAVID 6.7) [20 (link), 21 (link)] was used to characterize functional gene clusters regulated by microRNA target genes.
3
Profiling Serum miRNAs in Children from CAMP Cohort
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We performed small RNA sequencing (RNA-seq) on baseline serum samples from 492 children from CAMP. Serum samples were stored in freezers at −80°C at the Channing Division of Network Medicine. Recent work has shown that serum miRNAs are extremely stable over many years.16 (link),17 (link) Small RNA-seq libraries were prepared by using the Norgen Biotek Small RNA Library Prep Kit and sequenced on the Illumina NextSeq 500 platform. The ExceRpt pipeline18 (link) was used for quality control (QC) of the RNA-seq data. Mapped read counts less than 5 were filtered, and miRNAs with coverage of less than 50% of all subjects were removed. All samples passed QC for the number of mapped reads and total reads, indicating that satisfactory miRNA concentration was available. Using the DESeq2 R package,19 (link) we normalized reads by relative log2 expression. We used a guided principal component analysis algorithm for identification of batch effects in our data.20 (link) These miRNA data have previously been deposited into the Gene Expression Omnibus under the accession number GSE134897.
4
miRNA Sequencing and Analysis Protocol
5
Profiling miRNAs and vsiRNAs in Arabidopsis
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After quality control tests, the small RNA libraries were constructed by Small RNA Library Prep Kit for Illumina. Single-end sequencing was performed on Illumina HiSeq 2000 with read length of 50 bp. To analyze the known miRNAs in each library, the clean sequencing data were aligned to A. thaliana reference genome by Bowtie2. Known miRNAs were analyzed by miRDP61 (link) and differentially expressed miRNAs were acquired by DESeq2. For vsiRNAs analysis, the total sRNAs clean reads were mapped to TCV genome by Bowtie2. The completely mapped sRNAs seqences were termed as vsiRNAs that were generated from virus after infection.
6
MicroRNA Sequencing and Expression Analysis
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MicroRNAs were isolated from cells using the miRNeasy mini kit (Qiagen) according to the manufacturer's instructions. Sequencing was performed at the Medical Genomics Facility, Mayo Clinic, Rochester, MN. Briefly, microRNAs were sequenced using the NEBNext Small RNA library prep kit on an Illumina HiSeq 2000. The Illumina reads were trimmed of adapters with Cutadapt [25] ). Trimmed microRNA sequences greater than 17 nucleotides in length were then aligned to the mm9 reference genome and the miRBase v19 reference sequences using Bowtie [26] (link). Known microRNA expression and novel microRNA prediction were quantified using the miRDeep2 package [27] . Differential expression analysis was performed with the Bioconductor R package EdgeR [28] (link). Individual microRNAs were amplified by Taqman assays (Applied Biosystems) for mmu-miR-615-3p and has-miR-615-3p (P131015-004E01) and normalized to the expression of sno-202 (P131022-004H07) for mouse, and U47 (P140221-004G06) for human, to calculate the dCT. Relative expression was expressed as fold change using the ddCT method, as described [29] (link).
7
Profiling Epilepsy-Derived Small RNAs in EVs
8
Small RNA Sequencing of Asthmatic Children
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We used available small RNA sequencing of blood serum from 398 children with mild-to-moderate persistent asthma from the CAMP. Small RNA-seq libraries were prepared using the Norgen Biotek (Thorond, Canada) Small RNA Library Prep Kit and sequenced on the Illumina (San Diego, CA, USA) NextSeq 500 platform by Norgen Biotek. The exceRpt pipeline was employed for the QC of the RNA-seq data.17 (link) We excluded miRs with mapped read counts < 5 or with coverage < 50% of all subjects from the study. Using the DESeq2 R package,18 (link) we normalized reads by relative log expression, which has been shown to be a robust normalization method.19 (link) The small RNA sequencing was performed in 22 batches, which can introduce technical effects due to inconsistencies during preparation and handling. Guided principal components analysis (gPCA)20 (link) was used to check for batch effects on mapped read counts per sample, which did not detect a significant batch affect after data normalization (P = 0.371, Supplementary Fig. S1 ).
9
Profiling Serum miRNAs in Children from CAMP Cohort
10
miRNA Library Preparation and Sequencing
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Preparation of miRNA libraries and sequencing was performed as described by Koppers-Lalic et al. [28 (link),29 (link)]. After RNA isolation, samples were adjusted to have the same amount of total RNA concentration (500 pg in 7 μL). Libraries were prepared using the standard small-RNA Library Prep Kit for Illumina. To assess samples’ quality before sequencing, the concentration of the samples was determined using the Fragment Analyzer and all samples met the quality requirements. Sequencing was performed using the HiSeq 4000 instrument (Illumina, San Diego, CA, USA) with 150 bp paired-ends.
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